In the quantitative determination of the concentration of a compound in a sample using a chromatograph, such as the gas chromatograph (GC) or liquid chromatograph (LC), the calibration curve method is normally used. The calibration curve method is a quantitative determination method in which the concentration is calculated from an analysis result with reference to a previously created calibration curve. Examples of this technique include the external standard method and internal standard method.
In the external standard method, a standard sample containing an analyte is prepared. A plurality of standard samples with different concentrations of the analyte are prepared, and a measurement is performed for each standard sample to collect data. A chromatogram is created from the collected data, and a peak originating from the analyte is located on the chromatogram. The area (or height) of this peak is computed, and a calibration curve showing the relationship between the peak area value and the concentration is created. After the measurement of an unknown sample is performed, the area value of the peak originating from the analyte on the chromatogram obtained by the measurement is computed. By comparing this area value with the calibration curve, the concentration of the analyte in the unknown sample is calculated. By the external standard method, a highly accurate quantitative determination can be achieved as long as there is no influence of foreign substances or other unfavorable factors. However, the method inconveniently requires the standard sample to be prepared for each analyte and the calibration curve to be created based on the result of the measurement of each standard sample.
On the other hand, in the internal standard method, a fixed quantity of internal standard substance whose retention time is as close to the analyte as possible within the range where the internal standard substance can be separated from the analyte by a chromatograph (and additionally, by the difference in their mass-to-charge ratios if a mass spectrometer is used as the detector) is added to the standard sample for the creation of calibration curves, and a measurement for the sample is performed to collect data. On a chromatogram created from those data, both the peak originating from the internal standard substance and the peak originating from the substance for the creation of calibration curves appear. The areas (or heights) of these peaks are individually computed, and a calibration curve showing the relationship between the peak area ratio and the concentration is created. In the measurement of an unknown sample, the fixed quantity of the same internal standard substance is added to the unknown sample, and the peak area ratio is computed from the chromatogram obtained by the measurement of the sample. By comparing this area ratio with the calibration curve, the concentration of the analyte in the unknown sample is calculated. According to the internal standard method, it is possible to avoid measurement errors due to various factors, such as the variation in the amount of sample injected into the chromatograph or the vaporization of the sample solvent.
In the testing of residual agricultural chemicals or environmental pollutants the screening of drugs and poisons or other similar measurements, it s necessary to determine the quantities of tens of compounds, or even hundreds of compounds with different physical properties based on the result of a single measurement. For such a simultaneous multicomponent analysis, a gas chromatograph mass spectrometer (GC/MS) or liquid chromatograph mass spectrometer (LC/MS), both of which are capable of separating components according to their mass-to-charge ratios in addition to the temporal separation of the components by the chromatograph, is commonly used. In the case of the simultaneous multicomponent analysis, it is practically impossible to prepare a standard sample, perform a measurement and create a calibration curve for each of all of the analytes from the viewpoints of the cost and efficiency of the analysis. Accordingly, it is impractical to adopt the quantitative determination which employs the external standard method.
Even in the case of the quantitative determination employing the internal standard method, or the semi-quantitative determination, it is considerably difficult to prepare an internal standard substance for each analyte. A conventional and common procedure for addressing this problem is as follows: The plurality of analytes are divided into groups each of which includes analytes whose retention times are close to each other. For each group, an appropriate kind of compound, or more specifically, a physically and chemically stable compound which is as close to that group as possible in terms of retention time, is assigned as the internal standard substance, and this internal standard substance is used in the quantitative determination by the internal method.
Another factor to be considered is that certain kinds of analytes are easily desorbed or decomposed during the analysis; for example, such a situation can occur in a GC/MS depending on the condition of the injection-port insert provided at the inlet of the column, the column, the ion source or other devices. The internal standard substance which is physically and chemically stable barely undergoes such desorption or decomposition. Therefore, if such an internal standard substance is used in the quantitative determination employing the internal standard method, it is often the case that a variation of the quantitative value due to the aforementioned factor cannot be corrected. In particular, such a situation is noticeable in the simultaneous multicomponent analysis of agricultural chemicals or similar substances which vary in physical properties including the polarity. Such a variation in the quantitative value does not only occur due to the previously described factors which depend on the condition of the devices; it also frequently occurs due to the loss of a portion of the analyte as a result of a sample pretreatment operation, such as the extraction from the sample, purification, condensation or constant-volume sampling of the analyte.
As a technique for correcting the variation in the quantitative value due to the various aforementioned factor (particularly, those associated with the sample pretreatment operation), a surrogate method has conventionally been used. In the surrogate method, a substance having similar physical properties to the analyte is selected as the surrogate. A known amount of surrogate is added to the sample before this sample is pretreated. After the pretreatment operation is completed, the recovery percentage of the surrogate in the obtained sample is determined and the quantitative value is corrected on the premise that the analyte is also recovered by the same percentage (for example, see Patent Literature 1). The surrogate should preferably be a compound which is separable from the analyte and yet is as similar to the analyte as possible in terms of the physical properties. In general, a compound which is structurally identical to the analyte and is labeled by a stable isotope (typically, deuterium) is used.
Normally, the internal standard substance is added to the sample after the pretreatment operation. However, it is also common to utilize the surrogate, which is added before the pretreatment operation, as the internal standard substance to create a calibration curve and perform the quantitative determination by the internal standard method. This technique enables a highly accurate quantitative determination which reflects the loss of the compound that occurs in the stage of the sample pretreatment operation.
However, in the simultaneous multicomponent analysis, it is practically impossible to prepare one surrogate for each of the analytes whose number exceeds several hundreds. The technique of assigning the internal standard substances as used in the previously described normal mode of simultaneous multicomponent analysis may also be similarly applicable in the case of utilizing the surrogates as the internal standard substances; i.e. it may be possible to divide analytes into groups and assign an appropriate kind of surrogate to each group. However, it is difficult to properly make such an assignment.